Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface.

The association of DNA Ligase IV (Lig4) with XRCC4 is essential for repair of DNA double-strand breaks (DSBs) by Non-homologous end-joining (NHEJ) in humans. DSBs cytotoxicity is largely exploited in anticancer therapy. Thus, NHEJ is an attractive target for strategies aimed at increasing the sensitivity of tumors to clastogenic anticancer treatments. However the high affinity of the XRCC4/Lig4 interaction and the extended protein-protein interface make drug screening on this target particularly challenging. Here, we conducted a pioneering study aimed at interfering with XRCC4/Lig4 assembly. By Molecular Dynamics simulation using the crystal structure of the complex, we first delineated the Lig4 clamp domain as a limited suitable target. Then, we performed in silico screening of ~95,000 filtered molecules on this Lig4 subdomain. Hits were evaluated by Differential Scanning Fluorimetry, Saturation Transfer Difference-NMR spectroscopy and interaction assays with purified recombinant proteins. In this way we identified the first molecule able to prevent Lig4 binding to XRCC4 in vitro. This compound has a unique tripartite interaction with the Lig4 clamp domain that suggests a starting chemotype for rational design of analogous molecules with improved affinity.

Generation of a single chain antibody variable fragment (scFv) to sense selectively RhoB activation.

Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation.

Chemical synthesis and biological evaluation of triazole derivatives as inhibitors of InhA and antituberculosis agents.

A series of triazoles have been prepared and evaluated as inhibitors of InhA as well as inhibitors of Mycobacterium tuberculosis H(37)R(v). Several of these new compounds possess a good activity against InhA, particularly compounds 17 and 18 for which molecular docking has been performed. Concerning their activities against M. tuberculosis H(37)R(V) strain, two of them, 3 and 12, were found to be good inhibitors with MIC values of 0.50 and 0.25 µg/mL, respectively. Particularly, compound 12 presenting the best MIC value of all compounds tested (0.6 µM) is totally inactive against InhA.

Synthesis and biological activities of triazole derivatives as inhibitors of InhA and antituberculosis agents.

InhA, the enoyl reductase from the mycobacterial type II fatty acid biosynthesis pathway, is a target for the development of novel drugs against tuberculosis. We exploited copper-catalyzed [3+2] cycloaddition between alkynes and different azides to afford 1,4-disubstituted triazole or α-ketotriazole derivatives. Several compounds bearing a lipophilic chain mimicking the substrate were able to inhibit InhA. Among them, 1-dodecyl-4-phenethyl-1H-1,2,3-triazole displayed a minimum inhibitory concentration inferior to 2 µg/mL against Mycobacterium tuberculosis H37Rv.

Syntheses of functionalized biotin N-1' derivatives: new tools for the control of gene expression with small molecules.

The exceptionally high affinity of biotin toward avidin and streptavidin is at the basis of (strept)avidin-biotin biotechnology, which has numerous applications in life sciences. Recent biotin developments for in vivo and in vitro acylation of selective targeted protein and intein-mediated site specific protein biotinylation require the free biotin carboxyl function to covalently bind with the targeted protein. However, recently this carboxylic function has been used to substitute biotin with numerous ligands and flags. In the present work, we propose the N-1' labeling possibilities of biotin, keeping the valeric chain free. We describe liquid and solid-phase syntheses of functionalized biotin N-1' derivatives. Although the N-1' modification involves a two-log decrease in affinity, in vitro these molecules kept their high avidin affinity (around 10(-12) M) and the in vivo acylation ability of new biotin derivatives.